Gerhard Heinrich

An improved algorithm for nucleic acid secondary structure display

An improved algorithm for the display of nucleic acid secondary structures is presented. It is particularly suitable for large sequence segments and it automatically generates an aesthetically pleasing display of the structure with very limited overlap of strands. Structural similarities in different structures are conserved in the display thus greatly aiding structural homology comparisons. Using the algorithm, we illustrate the effect of ribosome translocation on the secondary structure of a rat neuropeptide messenger RNA.

Neuropeptide Y gene expression in PC12 cells and its regulation by nerve growth factor: a model for developmental regulation

Neuropeptide Y (NPY) is a 36 amino acid neuronal peptide which has previously been described within a subset of sympathetic neurons. The presence of the messenger RNA that encodes NPY (NPY mRNA) has been studied in the PC12 cell line, as this is commonly used as a model of neuronal differentiation. Low levels of the NPY mRNA were identified in resting naive cells. Levels were induced five-fold by the addition of nerve growth factor to the medium at a dose of 30 ng/ml. Lower doses of nerve growth factor had no effect on NPY mRNA. The effect was rapid in onset being apparent within 12 h and near maximal at 24 h. A smaller two-fold increase in NPY mRNA was observed in cells treated with epidermal growth factor (3 ng/ml) over the same time course but no effect was observed with fibroblast growth factor (3 ng/ml), bradykinin (10(-6) M) or dexamethasone (10(-6) M). These results indicate that NPY gene expression is regulated in PC12 cells at the level of NPY mRNA.

Production and characterization of biologically active recombinant human nerve growth factor.

Nerve growth factor (NGF) is required for the differentiation and maintenance of sympathetic and sensory neurons. In animal models, NGF prevents the death of septal and basal forebrain cholinergic neurons deprived of endogenous NGF, suggesting that NGF may be of benefit in neurodegenerative diseases of humans. However, little is known about NGF in human brain, partly because a sensitive assay for hNGF has been lacking. As a first step toward developing the tools for the study of NGF in humans, recombinant human NGF (rhNGF) was produced by expressing exon 4 of the human NGF gene in COS cells. The expression vector is driven by the adenovirus major late promoter and contains an SV40 origin of replication. NGF was secreted by transiently transfected cells. Conditioned medium was assayed with an enzyme immunoassay (EIA) that utilizes a monoclonal antibody (clone 27/21) against mouse beta-NGF, and contained 15 ng/ml of rhNGF. The rhNGF migrated as a dimer of 26-29 Kd on a gel permeation chromatography column, and stimulated neurite outgrowth and neuropeptide Y mRNA levels in PC12 cells. With optimization, the described expression system is capable of providing sufficient hNGF for research and therapeutic purposes.

Regulation of nerve growth factor gene expression in primary brain monolayer cultures: effects of dibutyryl cyclic AMP and sodium butyrate

Primary brain cultures composed of a heterogeneous population of cells, most of which reacted with anti-fibronectin and only few with anti-GFAP antiserum, were found to synthesize a mRNA indistinguishable from mouse submandibular gland nerve growth factor (NGF) mRNA in hybridization characteristics and electrophoretic behavior in agarose gels. The levels of this mRNA were increased 2.5-10 fold by 1 mM dibutyryl cyclic AMP (db-cAMP) and sodium butyrate within 24 h, but not by 10 microM forskolin, and were unaffected by serum in the culture medium. These observations demonstrate that brain cells synthesize a NGF mRNA in primary culture and that the butyrate moiety of db-cAMP enhances NGF gene expression in these cells, probably by a modification of chromatin structure in and around the NGF gene.

The Glucagon Genes

Glucagon is a peptide hormone of 29 amino acids produced and secreted by the A cells of the pancreatic islets (1). It is a member of a structurally related group of peptides that includes secretin (2), vasoactive intestinal peptide (3), gastric inhibitory peptide (4), and growth hormone releasing hormone (5) (Fig. 1). The secretion of glucagon is regulated by blood levels of glucose (6) and amino acids (7), as well as by a variety of hormonal stimuli (8). The action of glucagon on its target tissues, particularly the liver, is an important factor in protein and carbohydrate metabolism (9,10). Abnormal regulation of glucagon gene expression has been implicated in the pathogenesis of diabetes mellitus (11).

Evidence from molecular probe hybridization that glicentin and pancreatic glucagon are encoded by the same gene

SOLUBILIZATION AND PHYSICAL SEPARATION OF ACTIVE HIGHAND LOW-AFFINITY RECEPTORS FOR VASOACTIVE INTESTINAL PEPTIDE (VIP). S. Paul, S.I. Said. U. of Okla Health Sciences Center & VA Medical Center, Okla City, USA, Molecular events at the receptor for VIP are likely to be of importance in physiology and disease. As a first step in studying the molecular biology of the VIPreceptor, we have used the zwitterionic detergent CHAPS (12mM) to solubilize VIPrecep~9~s from guinea pig lung membranes. The receptor assay was done by incubating ~-~I-VIP (90-110pm; purified by reverse-phase HPLC) with the solubilized proteins (10-25 ~g) without and with excess (5 ~m) unlabeled VlP. Bound.YlP was separated with Dextran-Norit A and centrifugation. Specific binding of IzDI-VlP increased with increasing concentration of the solubilized receptors, was saturable with increasing time and was competitively displaced by increasing concentrations of unlabeled VIP (B~n=133pM). The soluhillzed lung proteins were fractionated on a high performance gelZfiltration column (Protelnpak 300 sw) in 3mM CHAPS. Two peaks of specific VIP-binding, of apparent molecular weight 370kDa and 25kDa, were obtained. Kd values for the 370kDa and 25kDa binding species were, respectively, 0.3nM and 971nM. The affinity of the 370kDa solubilized receptor was 9.7 fold higher than the high-affinity receptor on intact lung-membranes (Kd=2.9nM). Rechromatography of the 370kDa-speeies in 3mM CHAPS yielded a single, identicall~ elutlng VIP-binding peak. When the 370kDa receptor was chromatographed in 9mM CHAPS, 41% of the VIP-binding activity emerged as the 270kDa species and the rest as the 25kDa species. The high affinity 370kDa material is probably a molecular complex that dissociates upon prolonged exposure to 9mM CHAPS, and generates the 25kDa low-affinity receptor. The availability of VIP-receptors in active and soluble form permits their molecular characterization and studies on signal transduction and the functional role of the VIP-receptor in physiologic and disease processes.